Compounds for stabilizing ryanodine receptors from aberrant levels of calcium release

ABSTRACT

Disclosed herein are methods and compositions comprising compounds capable of normalizing neuronal calcium dyshomeostasis. Also disclosed are methods comprising these compounds for treating neuronal or neurological disorders, including Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, fronto-temporal dementia, Pick&#39;s disease, chronic traumatic encepholopathy, traumatic brain injury, stroke, cerebellar ataxia, multiple sclerosis, Down syndrome, and aging-related CNS disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/716,138, filed May 19, 2015, now U.S. Pat. No. 9,725,429, which is acontinuation-in-part of U.S. patent application Ser. No. 14/213,614,filed Mar. 14, 2014, now U.S. Pat. No. 9,102,645, which claims thebenefit of priority from U.S. Provisional Application No. 61/794,455,filed Mar. 15, 2013, which are incorporated by reference herein in itsentirety.

BACKGROUND

An effective drug regimen remains elusive for the treatment ofneurodegenerative diseases including Alzheimer's disease (AD),Parkinson's disease, Huntington's disease, fronto-temporal dementia, aswell as brain injury, such as traumatic brain injury (TBI) and relatedcognitive deficits, and CTE (chronic traumatic encephalopathy).

To date, multiple Phase III clinical trials for the treatment of AD andother neurodegenerative diseases have been unsuccessful, not necessarilybecause they did not engage their target, but the target may bedissociated from the desired therapeutic outcome, namely retention ofcognitive abilities. The majority of the clinical trials for ADtherapies have focused on amyloid precursor protein (APP) processing,and while amyloid levels were reduced in many of the clinical trials,there was no positive effect on memory performance. Furthermore,complications such as toxicity problems and worsened cognitive functionshave emerged (Golde et al., 2009, Science 324:603-604; Kerchner & Boxer,2010, Expert Opinion on Biological Therapy 10:1121-1130; Chakroborty andStutzmann, 2013 Dec. 6, Eur J Pharmacol., pii: S0014-2999(13)00883-2.doi: 10.1016/j.ejphar.2013.11.012 [Epub ahead of print]). A possiblereason why these compounds did not succeed is that they wereadministered too late after the cellular and synaptic pathologyoccurred, and clearing amyloid at this stage would not improve synapticdamage.

An alternative approach to treatment of AD and other neurodegenerativediseases is to target aberrant pathogenic calcium signaling cascades.Stabilization of calcium signaling targets a pathogenic mechanism thatis tied to many major features and risk factors of neurodegenerativediseases. Rather than targeting a single diagnostic endpoint, such asamyloid aggregation, this strategy aims to normalize a pathogenicaccelerant—namely, sustained calcium dyshomeostasis—that is linked toamyloid pathology, tau hyperphosphorylation, apoptosis, synapticpathophysiology, and memory deficits.

Calcium signaling in neurons is fundamental to numerous criticalfunctions, including gene transcription, cell death, synaptic integrity,synaptic plasticity, and memory encoding. For example, early increasesin endoplasmic reticulum (ER) calcium release through ryanodine receptor(RyR) channels occur in a host of AD models, and in cells from familialand sporadic AD patients. Notably, RyR isoform 2 (RyR2) expression isaltered in AD patients and mouse models as well. Neuronal calciumdyshomeostasis is linked to all the major risk factors,histopathological features, synaptic deficits, and cognitive impairmentsthat define AD; therefore, stabilizing ER calcium can broadly impact arange of AD-linked pathologies. Several recent studies have demonstratedthat treating neurons from AD mice with dantrolene, a clinicallyavailable RyR stabilizer, reduces amyloid deposition, improves memoryperformance, reverses intracellular calcium alterations, and normalizesRyR expression (Chakroborty and Stutzmann, 2013 Dec. 6, Eur JPharmacol., pii: S0014-2999(13)00883-2. doi:10.1016/j.ejphar.2013.11.012 [Epub ahead of print]; Oules et al., 2012,Journal of Neuroscience 32:11820-11834). Similar therapeutic effects arealso evident with models of traumatic brain injury (TBI). Chronicdantrolene treatment in TgCRND8 mice after exposure to a mild TBImarkedly reduces the amount of pathological tau phosphorylation (FIG.5). However, dantrolene may be pathogenic; chronic oral treatment (10+months) with dantrolene was found to increase amyloid pathology (Zhanget al., 2010, Journal of Neuroscience 30:8566-8580).

Thus, there is a need in the art for novel compounds capable ofstabilizing ryanodine receptor channels.

SUMMARY

The present disclosure provides certain advantages and advancements overthe prior art. In particular, the present disclosure providescompositions and methods comprising novel RyR-targeted small moleculecompounds. Treatment with the RyR channel stabilizers disclosed hereinnormalizes aberrant ER calcium signaling and preserves synapticfunctions toward treating neurodegenerative diseases, such asAlzheimer's disease, where RyR channel stabilizers reduce amyloidpathology.

Thus, one aspect the disclosure provides compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl or heteroaryl, each of which is optionally substituted with1, 2, 3, 4, or 5 R⁶ groups;

W is S or O;

Y is substituted (aryl)C₁₋₆ alkyl- or

where

Z is C₁₋₆ alkyl, benzyl, aryl, heteroaryl, or NR⁴R⁵, wherein alkyl,benzyl, aryl, or heteroaryl is optionally substituted with 1, 2, 3, 4,or 5 independently selected R⁷ groups;

R³ is hydrogen, or R³ together with Z optionally forms a heterocyclicring optionally substituted with one or more of R⁷ groups;

R¹ and R² are each independently selected from hydrogen, halo, CN,nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein each is optionally substituted at asuitable position with 1, 2, or 3 groups independently selected fromhalo, CN, hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino;

R⁴ and R⁵ are each independently selected from hydrogen, C₁₋₆ alkyl,C₁₋₆ haloalkyl, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di-C₁₋₄alkylaminosulfonylamino, and (aryl)-heteroaryl-CH═N—, or R⁴ and R⁵together with nitrogen to which they are attached forms a heterocyclicring, wherein each moiety is optionally substituted at a suitableposition with 1, 2, or 3 groups independently selected from halo, CN,hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino; and

R⁶ and R⁷ are each independently selected from halo, CN, nitro, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆alkylamino, di-C₁₋₄-alkylamino, carboxy, carbamyl, C₁₋₆ alkylcarbamyl,di(C₁₋₄ alkyl)carbamyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄alkylaminosulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino,di-C₁₋₄ alkylaminosulfonylamino, and oxo, wherein each is optionallysubstituted at a suitable position with 1, 2, or 3 groups independentlyselected from halo, CN, hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino;

provided that the compound is not1-{[5-(4-nitrophenyl)-2-furyl]methylideneamino} imidazolidine-2,4-dioneor 1-{[5-(4-bromophenyl)-2-furyl]methylideneamino}imidazolidine-2,4-dione.

In some embodiments, Ar is aryl optionally substituted with 1, 2, 3, 4,or 5 R⁶ groups.

In some embodiments, the disclosure provides compounds of formula Iwhere the compound is of formula II:

-   wherein R^(a1), R^(a2), and R^(a3) are independently selected from    hydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆    alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino,    carboxy, carbamyl, C₁₋₆ alkylcarbamyl, and di(C₁₋₄ alkyl)carbamyl.

In some embodiments, W is O. In some embodiments, R¹ and R² are bothhydrogen. In some embodiments, the disclosure provides compounds offormula I or II wherein Y is

where Z is C₁₋₆ alkyl, benzyl, aryl, heteroaryl, or NR⁴R⁵, whereinalkyl, benzyl, aryl, or heteroaryl is optionally substituted with 1, 2,3, 4, or 5 independently selected R⁷ groups; and R³ is hydrogen.

In some embodiments, Z is methyl or ethyl. In some embodiments, Z isbenzyl. In some embodiments, Z is pyridinyl. In some embodiments, Z isNR⁴R⁵. In some embodiments, R⁴ and R⁵ are hydrogen. In some embodiments,R⁴ and R⁵ together with nitrogen form piperazinyl ring optionallysubstituted with C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino

In some embodiments, the disclosure provides compounds of formula I orII wherein Z is C₁₋₆ alkyl, benzyl, aryl, heteroaryl, or NR⁴R⁵, whereinalkyl, benzyl, aryl, or heteroaryl is optionally substituted with 1, 2,3, 4, or 5 independently selected R⁷ groups; and R³ together with Zoptionally forms a heterocyclic ring optionally substituted with one ormore of R⁷ groups.

In some embodiments, the disclosure provides compounds of formula I,wherein the compounds are selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

The disclosure also provides synthetic intermediates that are useful inmaking the compounds of formula I or II. The disclosure also providesmethods of preparing compounds of the disclosure and the intermediatesused in those methods.

Another aspect of the disclosure provides for pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, solvent, adjuvant ordiluent and one or more compounds of formula I or II.

In another aspect, the disclosure provides methods for normalizingneuronal calcium dyshomeostasis in a subject comprising administering tothe subject an effective amount of one or more compounds of formula I orII. In some embodiments, the subject is a human subject.

In another aspect, the disclosure provides methods for treating aneurological or neurodegenerative disorder in a subject comprisingadministering to the subject an effective amount of one or morecompounds of formula I or II.

In another aspect, the disclosure provides compositions for treating aneurological or neurodegenerative disorder comprising one or morecompounds of formula I or II.

In another aspect, the disclosure provides uses of one or more compoundsof formula I or II for preparing compositions for treating aneurological or neurodegenerative disorder.

In some embodiments, the neurological or neurodegenerative disorder isAlzheimer's disease, Parkinson's disease, Huntington's disease,fronto-temporal dementia, Pick's disease, chronic traumaticencepholopathy, traumatic brain injury, stroke, cerebellar ataxia,multiple sclerosis, Down syndrome, or an aging-related CNS disorder. Insome embodiments, the neurological or neurodegenerative disorder isAlzheimer's disease. In some embodiments, the subject is a humansubject.

These and other features and advantages of the present invention will bemore fully understood from the following detailed description of theinvention taken together with the accompanying claims. It is noted thatthe scope of the claims is defined by the recitations therein and not bythe specific discussion of features and advantages set forth in thepresent description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentinvention can be best understood when read in conjunction with thefollowing drawings, in which:

FIG. 1 shows representative images of RyR-evoked Ca²⁺ responses withinthe soma (FIG. 1A) or dendrites (FIG. 1B) from a NonTg hippocampal CA1neuron. FIGS. 1C and 1D illustrate the same as above except in an ADneuron. These results demonstrate increased RyR-evoked Ca²⁺ release in3×Tg-AD neurons. FIG. 1E is a bar graph that shows RyR-evoked Ca²⁺responses averaged per compartment for NonTg light gray and AD dark grayneurons. In all compartments, the AD values are significantly greaterthan NonTg; p<0.05.

FIG. 2 shows reversal of AD pathology in AD mouse models after 4-weektreatment with dantrolene. Saline (Sal)-treated APP/PS1 mice havesignificantly increased ER calcium responses in the soma (FIG. 2A) anddendrites (FIG. 2B), whereas 4-week dantrolene treatment normalizes thiscalcium response, with no effect on the NonTg controls. Representativegrayscale 2-photon images are shown in FIG. 2C. As shown in FIG. 2D,beta amyloid levels, indicated by 4G8 immunostaining, are reduced by 45%in hippocampus and cortex of dantrolene-treated mice. FIG. 2Edemonstrates that synaptic integrity, measured by colocalization of pre-and post-synaptic markers (synaptophysin and PSD-95 immunostaining,respectively) with confocal microscopy, is restored in thedantrolene-treated AD mice. *=p<0.05

FIG. 3 shows normalization of RyR2 expression levels in AD transgenicmice after 4 weeks of dantrolene treatment. Bar graphs show relativemRNA expression levels of the RyR2 (FIG. 3A) and RyR3 (FIG. 3B) isoformsfrom the hippocampus of NonTg and AD-Tg mice (3×Tg-AD, shown; APP/PS1,not shown) treated with 0.9% saline or 10 mg/kg dantrolene. AD-Tg micetreated with dantrolene show normalized expression levels of RyR2compared to saline treated AD-Tg mice. *=p<0.05;

FIG. 4 shows reduction of phospho-tau levels in the hippocamus ofTG-CRND8 AD mice following chronic dantrolene (Ryanodex; 10 mg/kg IP; 4weeks) treatment. Immunostaining against phospo-tau with CP13 showswidespread staining in the dentate gyrus in saline-treated mice (4months of age) (FIG. 4A) but not in dantrolene-treated mice (FIG. 4B).Upon stabilization of RyR-calcium signaling with dantrolene, phospho-taustaining is significantly reduced. FIG. 4C is a histogram showingaveraged % of area with CP13 fluorescence staining in the dentate gyrus.*p<0.05. n=9 slices/3 animals/group.

FIG. 5 shows reduction of pathological phospho-tau levels resulting froma single traumatic brain injury (controlled cortical impact) in C57 micefollowing chronic dantrolene (Ryanodex; 10 mg/kg IP; 4 weeks) treatment.Immunostaining against phospo-tau with CP13 shows widespread staining inthe dentate gyrus in saline-treated mice (FIG. 5A) but not indantrolene-treated mice (FIG. 5B). Upon stabilization of RyR-calciumsignaling with dantrolene, CP13 phospho-tau staining is significantlyreduced. FIG. 5C is a histogram showing averaged % of area with CP13fluorescence staining in the dentate gyrus. *p<0.05. n=6-9 slices/3animals/group.

FIG. 6 shows the structures of RyR-stabilizing molecules, RD05 and RD14,which have comparable or superior effects on Ca²⁺ release and markedlyimproved solubility and CNS penetration compared with dantrolene.

FIGS. 7A and 7B are histograms showing maximal calcium responses incultured N2A cells upon strong stimulation with the RyR agonist,caffeine (10 mM). In FIG. 7A, the average maximal RyR-evoked calciumresponse is shown with the solid light gray bar, the darker gray bar toits right shows the reduced calcium response to the same agonist incells pre-incubated with the RyR channel stabilizer dantrolene(Ryanodex, 10 mM) for 30 minutes. Here, the evoked RyR-calcium responseis reduced by over 60%. The following (patterned) bars show cellsincubated in various RyR channel stabilizers disclosed herein (10 μM),and the majority of these show similar to enhanced calcium stabilizingproperties (dotted line represents the desired range of effects)compared to dantrolene. FIG. 7B demonstrates the same series ofexperiments conducted with additional sets of compounds. N=3-6 platesper compound; * p<0.05, one-way ANOVA.

FIG. 8 shows the effects of novel RyR-stabilizing compounds on neuronalcalcium signaling and electrophysiological membrane properties in3×Tg-AD mouse models. Hippocampal brain slices were exposed to the RyRagonist, caffeine, under control (ctrl) conditions or after incubationin dantrolene or one of the RyR channel stabilizing compounds disclosedherein (10 μM, 1 hour). RyR-evoked calcium responses (FIG. 8A) andvoltage-gated calcium responses (FIG. 8B) were subsequently measured inCA1 pyramidal neurons with 2-photon microscopy and whole cell patchclamp recordings. Resting membrane potential (RMP; FIG. 8C) and membraneinput resistance (Ri; FIG. 8D) were also measured under theseconditions. In FIG. 8A, the enhanced RyR-evoked calcium response in theAD neurons under control conditions were significantly reduced to withinphysiological levels with dantrolene, CK013, and CK017. Dashed lineindicates approximate desired level of the RyR-evoked calcium response.There were no significant differences in the voltage-gated calciumresponses (FIG. 8B), RMP (FIG. 8C), or Ri (FIG. 8D) upon incubation withthe various compounds. *p<0.05. n=4-7 neurons/group.

FIG. 9 shows normalization of RyR-evoked calcium responses in acutehippocampal brain slices from adult 3×Tg-AD mice following chronictreatment with RyR-channel stabilizers CK013 and CK017 (10 mg/kg, ip; 4weeks). Peak RyR-evoked calcium responses in CA1 hippocampal neuronswere evoked by bath application of 10 mM caffeine. The saline-treated3×Tg-AD mice generated significantly larger calcium responses relativeto the NonTg Saline treated mice and from the CK013 and CK017 treated3×Tg-AD mice. Calcium responses from the CK013 and CK017 drug-treated3×Tg-AD mice were not different from the control NonTg saline treatedmice. *=One-way ANOVA: F_((3,38))=60.6; p<0.001.

FIG. 10 illustrates complete Table 1, which provides structure-activityrelationships for compounds selected from the initial round ofsynthesis.

Skilled artisans will appreciate that elements in the Figures areillustrated for simplicity and clarity and have not necessarily beendrawn to scale. For example, the dimensions of some of the elements inthe Figures can be exaggerated relative to other elements to helpimprove understanding of the embodiment(s) of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

Before describing the present invention in detail, a number of termswill be defined. As used herein, the singular forms “a”, “an”, and “the”include plural referents unless the context clearly dictates otherwise.For example, reference to a “protein” means one or more proteins.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that can or cannot be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that can be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation can vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

When trade names are used, it is intended to independently include thetrade name product formulation, the generic drug and the activepharmaceutical ingredient(s) of the trade name product.

Terms used herein may be preceded and/or followed by a single dash, “—”,or a double dash, “═”, to indicate the bond order of the bond betweenthe named substituent and its parent moiety; a single dash indicates asingle bond and a double dash indicates a double bond. “

” means a single or double bond. In the absence of a single or doubledash it is understood that a single bond is formed between thesubstituent and its parent moiety; further, substituents are intended tobe read “left to right” unless a dash indicates otherwise. For example,C₁-C₆alkoxycarbonyloxy and —OC(O)C₁-C₆alkyl indicate the samefunctionality; similarly arylalkyl and -alkylaryl indicate the samefunctionality.

When chemical structures are depicted or described, unless explicitlystated otherwise, all carbons are assumed to have hydrogen substitutionto conform to a valence of four. For example, in the structure on theleft-hand side of the schematic below there are nine hydrogens implied,as depicted in the right-hand structure. Sometimes a particular atom ina structure is described in textual formula as having a hydrogen orhydrogens as substitution (expressly defined hydrogen), for example,—CH₂CH₂—. It is understood by one of ordinary skill in the art that theaforementioned descriptive techniques are common in the chemical arts toprovide brevity and simplicity to description of otherwise complexstructures.

The term “alkoxy” as used herein, means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

The term “alkyl” as used herein, means a straight or branched chainhydrocarbon containing from 1 to 20 carbon atoms unless otherwisespecified. Representative examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl. The term “alkylene” refers to a divalent alkyl group, wherealkyl is as defined herein.

The term “aryl,” as used herein, means a phenyl (i.e., monocyclic aryl),or a bicyclic ring system containing at least one phenyl ring or anaromatic bicyclic ring containing only carbon atoms in the aromaticbicyclic ring system, or a polycyclic ring system containing at leastone phenyl ring. The bicyclic aryl can be azulenyl, naphthyl, or aphenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or amonocyclic heterocyclyl. The bicyclic aryl is attached to the parentmolecular moiety through any carbon atom contained within the phenylportion of the bicyclic system, or any carbon atom with the napthyl orazulenyl ring.

The terms “cyano” and “nitrile” as used herein, mean a —CN group.

The term “halo” or “halogen” as used herein, means —Cl, —Br, —I or —F.

The terms “haloalkyl” and “haloalkoxy” refer to an alkyl or alkoxygroup, as the case may be, which is substituted with one or more halogenatoms.

The term “heteroaryl,” as used herein, means a monocyclic heteroaryl ora bicyclic ring system containing at least one heteroaromatic ring. Themonocyclic heteroaryl can be a 5 or 6 membered ring. The 5 membered ringconsists of two double bonds and one, two, three or four nitrogen atomsand optionally one oxygen or sulfur atom. The 6 membered ring consistsof three double bonds and one, two, three or four nitrogen atoms. The 5or 6 membered heteroaryl is connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within theheteroaryl. The bicyclic heteroaryl consists of a monocyclic heteroarylfused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, amonocyclic heterocyclyl, or a monocyclic heteroaryl. The bicyclicheteroaryl may be attached through either cyclic moiety (e.g., eitherthrough heteroaryl or through phenyl.) Representative examples ofheteroaryl include, but are not limited to, furyl, imidazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, triazinyl, benzimidazolyl, benzofuranyl,benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl,cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl,furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl,quinolinyl, or purinyl.

The term “heterocyclyl” as used herein, means a monocyclic heterocycleor a bicyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or7 membered ring containing at least one heteroatom independentlyselected from the group consisting of O, N, and S where the ring issaturated or unsaturated, but not aromatic. The 3 or 4 membered ringcontains 1 heteroatom selected from the group consisting of O, N and S.The 5 membered ring can contain zero or one double bond and one, two orthree heteroatoms selected from the group consisting of O, N and S. The6 or 7 membered ring contains zero, one or two double bonds and one, twoor three heteroatoms selected from the group consisting of O, N and S.The bicyclic heterocycle is a monocyclic heterocycle fused to either aphenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocycle, or a monocyclic heteroaryl. The bicyclic heterocycle may beattached through either cyclic moiety (e.g., either through heterocycleor through phenyl.) Representative examples of heterocycle include, butare not limited to, aziridinyl, diazepanyl, 1,3-dioxanyl,1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl,imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl,isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl,oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl,pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl,tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl,thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, trithianyl,2,3-dihydrobenzofuran-2-yl, and indolinyl.

The phrase “one or more” substituents, as used herein, refers to anumber of substituents that equals from one to the maximum number ofsubstituents possible based on the number of available bonding sites,provided that the above conditions of stability and chemical feasibilityare met. Unless otherwise indicated, an optionally substituted group mayhave a substituent at each substitutable position of the group, and thesubstituents may be either the same or different. As used herein, theterm “independently selected” means that the same or different valuesmay be selected for multiple instances of a given variable in a singlecompound.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. One of ordinary skill in the art would understand that withrespect to any molecule described as containing one or more optionalsubstituents, only sterically practical and/or synthetically feasiblecompounds are meant to be included. “Optionally substituted” refers toall subsequent modifiers in a term, unless stated otherwise.

The term “substituted”, as used herein, means that a hydrogen radical ofthe designated moiety is replaced with the radical of a specifiedsubstituent, provided that the substitution results in a stable orchemically feasible compound. The term “substitutable”, when used inreference to a designated atom, means that attached to the atom is ahydrogen radical, which can be replaced with the radical of a suitablesubstituent.

Disclosed herein are compounds capable of normalizing neuronal calciumdyshomeostasis, of stabilizing RyR channels, and/or of normalizingaberrant neuronal calcium signaling, such as aberrant neuronalendoplasmic reticulum (ER) calcium signaling. In some embodiments, thedisclosure provides methods and compositions comprising these compoundsfor treating neuronal or neurodegenerative disorders, such asAlzheimer's disease.

As used herein, the term “normalize” refers to returning an aberrant orabnormal biological quantity to normal or typical levels, such as levelstypical of healthy neurons; alternatively, “normalize” refers toreturning a state of disregulation or dyshomeostasis to a regulatedstate or a state of homeostasis, such as in healthy, typical, ornormally functioning neurons.

As used herein, the term “calcium dyshomeostasis” refers to thedisregulation of intracellular calcium in a cell, for example a neuron,for example, due to changes in the permeability of calcium channels,such as RyR channels.

The calcium ion (Ca²⁺) is the main chemical messenger that helpstransmit synaptic activity and depolarization status to the biochemicalsystems of a neuron. Thus, regulating Ca²⁺ levels-maintaining “calciumhomeostasis”—is a critical process in neurons, which rely on extensiveand intricate Ca²⁺ signaling pathways. As used herein, the term “calciumsignaling” refers to all of the Ca²⁺ signaling that occurs withinneurons.

As used herein, the term “stabilize” when used with respect to an ionchannel,” such as an RyR channel, refers to modulating the flow or fluxof ions through the channel, for example decreasing an abnormally highion flux or increasing an abnormally low ion flux. In some embodimentsof the methods and compositions disclosed herein, stabilizing an RyRchannel with an RyR-channel stabilizing compound decreases the Ca²⁺ fluxthrough the channel, such that calcium dyshomeostasis or disregulationin a neuron is normalized and calcium homeostasis is restored.

Of particular relevance to the compositions and methods disclosed hereinis the increased ER-Ca²⁺ release mediated by ryanodine receptors.Ryanodine receptors (RyRs) are a class of intracellular calcium channelslocalized to the endoplasmic reticulum (ER) in various forms ofexcitable animal tissue like muscles and neurons. RyRs are activated byCa²⁺ itself in a regenerative process termed Ca²⁺-induced Ca²⁺ release(CICR).

In Alzheimer's disease (AD) models, the threshold for inducing CICR issignificantly lowered, such that normally innocuous Ca²⁺ entry triggeredby synaptic stimulation or N-methyl-D-aspartate receptor (NMDAR)activation will now trigger an aberrant CICR response. Not only is theamount of RyR-Ca²⁺ released abnormally high, but the cellularcompartments where it occurs is abnormal, such that greatly exaggeratedRyR-evoked Ca²⁺ release occurs in synaptic compartments such as distaldendrites and spine heads; normally, RyR-Ca²⁺ release is observed in lowlevels in these regions in NonTg neurons (see FIG. 1). The synapticcalcium dyshomeostasis disrupts synaptic transmission and plasticityencoding, and can drive dendritic spine loss.

Neuronal calcium dyshomeostasis is likely a central component of ADwhich drives early pathogenesis, and sustains amyloid pathology insporadic AD. Furthermore, the dysregulation in synaptic calcium signalsobserved in the AD models is closely associated with synapticdysfunction and structural impairment. The RyR is directly implicated inaccelerating amyloid deposition (Querfurth et al., 1997, Journal ofNeurochemistry 69:1580-1591), and in human brain studies, elevated RyRsare observed in MCI and AD patients, and are linked to cognitive declineand synaptic pathology (Kelliher et al., 1999, Neuroscience 92:499-513;Galeotti et al., 2008, Learning & Memory 15:315-323). Increased RyR2isoform expression was also observed in human MCI patients, and inseveral AD mouse models (Chakroborty et al., 2009, J. Neurosci.29(30):9458-70; Goussakov et al., 2010, J Neurosci 30:12128-12137; Brunoet al., 2012, Neurobiology of Aging 33:1001.e1001-1001.e106.; Antonellet al., 2013, Neurobiology of Aging 34:1772-1778), and others have foundRyR3 upregulation at later disease stages coincident with Aβ₁₋₄₂aggregation (Supnet et al., 2006, Journal of Biological Chemistry 281:38440-38447).

In some embodiments, the methods and compositions disclosed hereincomprise compounds that allosterically modulate the RyR channel, ratherthan non-selectively block activity as per conventional pharmacologicalantagonists. In this manner, the disclosed compounds normalize aberrantCa²⁺ signals and halt or prevent pathogenesis caused by calciumdyshomeostasis while leaving functional Ca²⁺ signals intact.

There are multiple isoforms of ryanodine receptors: ryanodine receptorisoform 1 (RyR1) is primarily expressed in skeletal muscle; ryanodinereceptor isoform 2 (RyR2) is expressed primarily in myocardium (heartmuscle), but is also highly expressed in the hippocampus, as discussedbelow; ryanodine receptor isoform 3 (RyR3) is expressed more widely, butespecially in the brain.

A particular target of interest for the methods and compositionsdisclosed herein is the RyR2 isoform, which is highly expressed inhippocampus, is involved in memory encoding, and is upregulated early inAD mouse models and human MCI patients. However, RyR2 is the primarycardiac isoform, and systemically ‘blocking’ this channel may generateundesirable off-target effects. Therefore, in some embodiments, thedisclosure provides compounds with higher affinity for the RyR2 isoformcompared to other isoforms.

In some embodiments, the disclosed RyR2-stabilizing compounds causereduction in the rate of cognitive decline in early and mid-stage ADpatients, along with a reduction in amyloid deposition,hyperphosphorylated tau, and a reduced conversion from MCI to AD. Theseeffects are likely due to preservation of synaptic structure andsynaptic plasticity that is compromised under conditions of sustainedcalcium dysregulation, such as seen during AD pathogenesis.

As the disclosed compounds are designed to stabilize RyR channel releaseproperties through allosteric modulation of the RyR channelphosphorylation and oxidation sites, and do not serve as classicalantagonists which block or reduce channel activity, there are likely noprofound side effects on cardiac function (cardiac myocytes expressRyR2). Rather, the compounds disclosed herein maintain normalphysiological function rather than indiscriminately block calciumreleased from RyR channels.

In some embodiments, pharmaceutical compositions comprising thedisclosed RyR-stabilizing compounds are orally available and takendaily. In some embodiments, the compounds and compositions disclosedherein are administered subcutaneously, and in some embodiments areadministered every other day. In some embodiments, the compounds'effects last weeks to months. In other embodiments, the compounds'effects last over a shorter period, such as when the calciumdyshomeostasis is downstream of a pre-existing pathology. However, inother embodiments, such as when RyR-calcium dysregulation is the primaryaccelerant of a feed-forward pathway, the compositions and compoundsdisclosed herein have long-term therapeutic effects.

As the person of ordinary skill in the art will appreciate, any of thecompounds disclosed herein can be provided as a derivative or prodrug,depending, e.g., on the desired end properties of the compositions andmethods. For example, RyR channel stabilizers may be modified with asuitable prodrug group that metabolizes or otherwise transforms underconditions of use to yield an RyR channel stabilizer. Derivatives of RyRchannel stabilizers to be used for the compositions and methods of thepresent disclosure are within the skill of the person skilled in the artusing routine trial and experimentation.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered.

In some embodiments, the active ingredients of the compositions andmethods disclosed herein are formulated as a pharmaceutically acceptablesalt. As used herein, the term “pharmaceutically acceptable salt”includes acid and base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like, as well as organic acids such as aceticacid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like.

“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Exemplary salts are the ammonium, potassium, sodium, calcium,and magnesium salts. Salts derived from pharmaceutically acceptableorganic non-toxic bases include, but are not limited to, salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplaryorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline, and caffeine. (See, forexample, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977; 66:1-19 which is incorporated herein by reference.)

In one aspect, the disclosure provides compounds of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl or heteroaryl, each of which is optionally substituted with1, 2, 3, 4, or 5 R⁶ groups;

W is S or O;

Y is substituted (aryl)C₁₋₆ alkyl- or

where

Z is C₁₋₆ alkyl, benzyl, aryl, heteroaryl, or NR⁴R⁵, wherein alkyl,benzyl, aryl, or heteroaryl is optionally substituted with 1, 2, 3, 4,or 5 independently selected R⁷ groups;

R³ is hydrogen, or R³ together with Z optionally forms a heterocyclicring optionally substituted with one or more of R⁷ groups;

R¹ and R² are each independently selected from hydrogen, halo, CN,nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein each is optionally substituted at asuitable position with 1, 2, or 3 groups independently selected fromhalo, CN, hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃ alkylamino;

R⁴ and R⁵ are each independently selected from hydrogen, C₁₋₆ alkyl,C₁₋₆ haloalkyl, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di-C₁₋₄alkylaminosulfonylamino, and (aryl)-heteroaryl-CH═N—, or R⁴ and R⁵together with nitrogen to which they are attached forms a heterocyclicring, wherein each moiety is optionally substituted at a suitableposition with 1, 2, or 3 groups independently selected from halo, CN,hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino; and

R⁶ and R⁷ are each independently selected from halo, CN, nitro, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆alkylamino, di-C₁₋₄-alkylamino, carboxy, carbamyl, C₁₋₆ alkylcarbamyl,di(C₁₋₄ alkyl)carbamyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄alkylaminosulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino,di-C₁₋₄ alkylaminosulfonylamino, and oxo, wherein each is optionallysubstituted at a suitable position with 1, 2, or 3 groups independentlyselected from halo, CN, hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino;

provided that the compound is not1-{[5-(4-nitrophenyl)-2-furyl]methylideneamino} imidazolidine-2,4-dioneor 1-{[5-(4-bromophenyl)-2-furyl]methylideneamino}imidazolidine-2,4-dione.

The disclosure also provides synthetic intermediates that are useful inmaking the compounds of formula II. The disclosure also provides methodsof preparing compounds of the disclosure and the intermediates used inthose methods.

Another aspect of the disclosure provides for pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, solvent, adjuvant ordiluent and one or more compounds of formula I.

The disclosure also provides compounds of formula I, wherein Ar is aryloptionally substituted with 1, 2, 3, 4, or 5 R⁶ groups.

Particular embodiments based on formula I include those where Ar isphenyl optionally substituted with 1, 2, or 3 R⁶ groups. Otherembodiments provide compounds where R⁶ is independently selected fromhalo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, and di(C₁₋₄ alkyl)carbamyl. For example,Ar may be phenyl, 4-chlorophenyl, 3-nitrophenyl, 2-methoxyphenyl, and2,3-dimethyl-4-nitrophenyl.

Other particular embodiments provide for compounds where Ar isheteroaryl optionally substituted with 1, 2, 3, 4, or 5 R⁶ groups.

Particular embodiments based on formula I include those where thecompound is of formula II:

wherein R^(a1), R^(a2), and R^(a3) are independently selected fromhydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino,carboxy, carbamyl, C₁₋₆ alkylcarbamyl, and di(C₁₋₄ alkyl)carbamyl; andR¹, R², W, and Y are as defined herein. Other embodiments provide forcompounds of formula II where R^(a1), R^(a2), and R^(a3) areindependently selected from hydrogen, halo, nitro, hydroxy, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy. In yet anotherembodiment, R^(a1), R^(a2), and R^(a3) are independently selected fromhydrogen, halo, nitro, and C₁₋₆ alkoxy.

Particular embodiments based on formula I or II and any precedingembodiment include those where W is O.

Other particular embodiments based on formula I or II and any precedingembodiment include those where W is S.

Embodiments based on formula I or II and any preceding embodimentinclude those where R¹ and R² are each independently selected fromhydrogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,amino, C₁₋₆ alkylamino, and di-C₁₋₄-alkylamino. Other embodimentsprovide for compounds of formula I or II where R¹ and R² are bothhydrogen. In one embodiment, one of R¹ and R² is hydrogen are the otheris C₁₋₆ alkyl.

Particular embodiments based on formula I or II and any precedingembodiment include those where Y is (aryl)C₁₋₆ alkyl-. One embodimentprovides for compounds where Y is benzyl. In another embodiment, Y is1-phenyl-ethyl-.

Particular embodiments based on formula I or II and any precedingembodiment include those where Y is

where

-   Z is C₁₋₆ alkyl, benzyl, aryl, heteroaryl, or NR⁴R⁵, wherein alkyl,    benzyl, aryl, or heteroaryl is optionally substituted with 1, 2, 3,    4, or 5 independently selected R⁷ groups; and-   R³ is hydrogen.

This embodiment provides for compounds wherein Z is C₁₋₆ alkyl, benzyl,aryl, or heteroaryl, each optionally substituted with 1, 2, 3, 4, or 5independently selected R⁷ groups. For example, in one embodiment, Z maybe C₁₋₆ alkyl, such as methyl or ethyl. In another exemplary embodiment,Z is benzyl. Other embodiments provide for compounds where Z is aryl orheteroaryl, each optionally substituted with 1, 2, 3, 4, or 5independently selected R⁷ groups. In one exemplary embodiment, Z is aryloptionally substituted with 1, 2, or 3 R⁷ groups. In another exemplaryembodiment, Z is phenyl optionally substituted with 1, 2, or 3 R⁷groups. Other embodiments provide for compounds where Z is heteroaryloptionally substituted with 1 or 2 R⁷ groups. In one exemplaryembodiment, Z is pyridinyl.

This embodiment also provides for compounds wherein Z is NR⁴R⁵.

In one embodiment, R⁴ and R⁵ are each independently selected fromhydrogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, amino, C₁₋₆ alkylamino,di-C₁₋₄-alkylamino, carboxy, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄alkyl)carbamyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄alkylaminosulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino,di-C₁₋₄ alkylaminosulfonylamino, and (aryl)-heteroaryl-CH═N—, whereineach optionally substituted at a suitable position with 1, 2, or 3groups independently selected from halo, CN, hydroxy, C₁₋₃ alkyl, C₁₋₃alkoxy, amino, C₁₋₃ alkylamino, and di-C₁₋₃-alkylamino. Certainembodiments provide for compounds where R⁴ and R⁵ are hydrogen. Otherembodiments provide for compounds where R⁴ is hydrogen, and R⁵ is C₁₋₆alkyl.

In another embodiment, R⁴ and R⁵ together with nitrogen to which theyare attached optionally forms a heterocyclic ring optionally substitutedat a suitable position with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino,and di-C₁₋₃-alkylamino. Other embodiments provide for compounds where R⁴and R⁵ together with nitrogen form piperazinyl ring optionallysubstituted with C₁₋₃ alkyl, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino.

Particular embodiments based on formula I or II and any precedingembodiment include those where Y is

where

-   Z is C₁₋₆ alkyl, benzyl, aryl, heteroaryl, or NR⁴R⁵, wherein alkyl,    benzyl, aryl, or heteroaryl is optionally substituted with 1, 2, 3,    4, or 5 independently selected R⁷ groups; and-   R³ together with Z optionally forms a heterocyclic ring optionally    substituted with one or more of R⁷ groups.

This embodiment provides for compounds R³ together with Z optionallyforms imidazolidine-2,5-dionyl.

In some embodiments, the compound of formula (I) is selected from thecompounds shown in Tables 1 and 2 or pharmaceutically acceptable saltsthereof.

In another aspect, the invention provides pharmaceutical compositionscomprising one or more compounds of formula (I) and a pharmaceuticallyacceptable carrier, excipient, or diluent. Administration of thecompounds of the invention, or their pharmaceutically acceptable salts,in pure form or in an appropriate pharmaceutical composition, can becarried out via any of the accepted modes of administration or agentsfor serving similar utilities. Thus, administration can be, for example,orally, nasally, parenterally (intravenous, intramuscular, orsubcutaneous), topically, transdermally, intravaginally, intravesically,intracistemally, rectally, or via urethral, ocular intratumoral,intraventricular, intrathecal, pulmonary and irrigation method, in theform of solid, semi-solid, lyophilized powder, or liquid dosage forms,such as, for example, tablets, suppositories, pills, soft elastic andhard gelatin capsules, powders, solutions, suspensions, or aerosols, orthe like, preferably in unit dosage forms suitable for simpleadministration of precise dosages.

The compositions will include a conventional pharmaceutical carrier orexcipient and a compound of the invention as the/an active agent, and,in addition, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, etc. Compositions of the invention may be used incombination with anticancer or other agents that are generallyadministered to a patient being treated for cancer or other disorder.Adjuvants include preserving, wetting, suspending, sweetening,flavoring, perfuming, emulsifying, and dispensing agents. Prevention ofthe action of microorganisms can be ensured by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,for example sugars, sodium chloride, and the like. Prolonged absorptionof the injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

If desired, a pharmaceutical composition of the invention may alsocontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, antioxidants, and the like,such as, for example, citric acid, sorbitan monolaurate, triethanolamineoleate, butylalted hydroxytoluene, etc. The dosage form can be designedas a sustained release or timed release.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), dextrose, mannitol,polyvinylpyrrolidone, gelatin, hydroxycellulose, acacia, suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. The liquid formulation can be buffered,isotonic solution.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose,mannitol, and silicic acid, (b) binders, as for example, cellulosederivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose,and gum acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, croscarmellose sodium, complexsilicates, and sodium carbonate, (e) solution retarders, as for exampleparaffin, (f) absorption accelerators, as for example, quaternaryammonium compounds, (g) wetting agents, as for example, cetyl alcohol,and glycerol monostearate, magnesium stearate and the like, (h)adsorbents, as for example, kaolin and bentonite, and (i) lubricants, asfor example, talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents.

Solid dosage forms as described above can be prepared with coatings andshells, such as enteric coatings and others well known in the art. Theymay contain pacifying agents, and can also be of such composition thatthey release the active compound or compounds in a certain part of theintestinal tract in a delayed manner. Examples of embedded compositionsthat can be used are polymeric substances and waxes. The activecompounds can also be in microencapsulated form, if appropriate, withone or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Suchdosage forms are prepared, for example, by dissolving, dispersing, etc.,a compound(s) of the invention, or a pharmaceutically acceptable saltthereof, and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose, glycerol, ethanol and thelike; solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide; oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan; or mixtures of these substances, and the like, to thereby forma solution or suspension.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions for rectal administrations are, for example, suppositoriesthat can be prepared by mixing the compounds of the present inventionwith for example suitable non-irritating excipients or carriers such ascocoa butter, polyethyleneglycol or a suppository wax, which are solidat ordinary temperatures but liquid at body temperature and therefore,melt while in a suitable body cavity and release the active componenttherein.

Dosage forms for topical administration of a compound of this inventioninclude ointments, powders, sprays, and inhalants. The active componentis admixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, eye ointments, powders, and solutionsare also contemplated as being within the scope of this invention.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable compositions will contain about 0.01% toabout 99.99% by weight of one or more compounds of the invention, or apharmaceutically acceptable salt thereof, and 99.99% to 0.01% by weightof a suitable pharmaceutical excipient. In one example, the compositionwill be between about 0.5% and about 75% by weight of a compound of theinvention, or a pharmaceutically acceptable salt thereof, with the restbeing suitable pharmaceutical excipients.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton,Pa., 1990). The composition to be administered will, in any event,contain a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof, for treatmentof a disease-state in accordance with the teachings of this invention.

In another aspect, the disclosure provides methods for normalizingneuronal calcium dyshomeostasis in a subject comprising administering tothe subject one or more compounds of formula (I). In some embodiments,the subject is a human subject.

In another aspect, the disclosure provides methods for treating aneurological or neurodegenerative disorder in a subject comprisingadministering to the subject one or more compounds of formula (I). Insome embodiments, the neurological or neurodegenerative disorder isAlzheimer's disease, Parkinson's disease, Huntington's disease,fronto-temporal dementia, Pick's disease, chronic traumaticencepholopathy, traumatic brain injury, stroke, cerebellar ataxia,multiple sclerosis, Down syndrome, or an aging-related CNS disorder. Insome embodiments, the neurological or neurodegenerative disorder isAlzheimer's disease. In some embodiments, the subject is a humansubject.

The compounds of the invention, or their pharmaceutically acceptablesalts, are administered in an “effective amount” or “therapeuticallyeffective amount” which will vary depending upon a variety of factorsincluding the activity of the specific compound employed, the metabolicstability and length of action of the compound, the age, body weight,general health, sex, diet, mode and time of administration, rate ofexcretion, drug combination, the severity of the particulardisease-states, and the host undergoing therapy. The compounds of thepresent invention can be administered to a patient at dosage levels inthe range of about 70 to about 1400 mg per day. For a normal human adulthaving a body weight of about 70 kilograms, a dosage in the range ofabout 1 to about 20 mg per kilogram of body weight per day is anexample. The specific dosage used, however, can vary. For example, thedosage can depend on a number of factors including the requirements ofthe patient, the severity of the condition being treated, and thepharmacological activity of the compound being used. The determinationof optimum dosages for a particular patient is well known to one ofordinary skill in the art.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of theinvention, and various uses thereof. They are set forth for explanatorypurposes only, and are not to be taken as limiting the invention.

The preparation of the compounds of the disclosure is illustratedfurther by the following examples, which are not to be construed aslimiting the disclosure in scope or spirit to the specific proceduresand compounds described in them. In all cases, unless otherwisespecified, the column chromatography is performed using a silica gelsolid phase.

Those having skill in the art will recognize that the starting materialsand reaction conditions may be varied, the sequence of the reactionsaltered, and additional steps employed to produce compounds encompassedby the present disclosure, as demonstrated by the following examples.Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Starting materials can be obtained from commercial sources or preparedby well-established literature methods known to those of ordinary skillin the art. The reactions are performed in a solvent appropriate to thereagents and materials employed and suitable for the transformationsbeing effected. It will be understood by those skilled in the art oforganic synthesis that the functionality present on the molecule shouldbe consistent with the transformations proposed. This will sometimesrequire a judgment to modify the order of the synthetic steps or toselect one particular process scheme over another in order to obtain adesired compound of the disclosure.

In some cases, protection of certain reactive functionalities may benecessary to achieve some of the above transformations. In general, theneed for such protecting groups as well as the conditions necessary toattach and remove such groups will be apparent to those skilled in theart of organic synthesis. An authoritative account describing the manyalternatives to the trained practitioner are J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London and NewYork 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups inOrganic Synthesis”, Third edition, Wiley, New York 1999, in “ThePeptides”; Volume 3 (editors: E. Gross and J. Meienhofer), AcademicPress, London and New York 1981, in “Methoden der organischen Chemie”,Houben-Weyl, 4.sup.th edition, Vol. 15/I, Georg Thieme Verlag, Stuttgart1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

Example 1: Materials and Methods

Brain Slice Ca²⁺ Signaling and Electrophysiology.

3×Tg-AD, TgCRND8, PS1/APP, and nontransgenic (NonTg) J29/C57BL6 controlmice were bred in-house. Adult male and female mice 3-5 months old wereused. Animals were cared for and used in accordance with protocolsapproved by the Rosalind Franklin University of Medicine and ScienceAnimal Care and Use Committee. Hippocampal brain slices (300 μm) wereprepared as previously described (Briggs et al., Neurobiol. Aging, 2013,34:1632-1643). Mice were anesthetized with halothane, decapitated, andthe brain removed into ice-cold sucrose cutting solution (200 mMsucrose, 1.5 mM KCl, 0.5 mM CaCl₂, 4.0 mM MgCl₂, 1.0 mM KH₂PO₄, 25 mMNaHCO₃, 10 mM Na-ascorbate, and 20 mM dextrose, equilibrated with 95%O₂/5% CO₂). Horizontal hippocampal slices were prepared in a CamdenInstruments vibratome with the chamber filled with ice-cold sucrosecutting solution and then transferred to and maintained in standardartificial cerebrospinal fluid (aCSF; 130 mM NaCl, 2.5 mM KCl, 2.0 mMCaCl₂, 1.2 mM MgSO4, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, and 10 mM dextrose[305-310 mOsm], equilibrated with 95% O₂/5% CO₂, pH 7.3-7.4) at 32° C.for at least 1 hour before use. Whole-cell patch-clamp recordings wereconducted at room temperature (23° C.) in continuously superfused aCSF(1.5-2.0 mL/min). Patch pipettes (5-7 MO) were filled with intracellularsolution (135 mM K-gluconate, 2.0 mM MgCl₂, 4.0 mM Na₂-ATP, 0.4 mMNa-GTP, 10 mM Na-phosphocreatine, 10 mM HEPES adjusted to pH 7.3 withKOH) plus 50 μM bis-fura-2 hexapotassium (Life Technologies) asfluorescent Ca²⁺ sensor. Hippocampal CA1 pyramidal neurons wereidentified visually via infrared differential interference contrast(IR-DIC) optics, and electrophysiologically by their passive membraneproperties and spike frequency accommodation. Membrane potentials wereobtained in current-clamp mode, acquired at 10 kHz with a Digidata 1322A-D converter and Multiclamp 700B amplifier, and recorded and analyzedusing pClamp 10.2 (Molecular Devices). Minianalysis 6.0.7 (Synaptosoft,Fort Lee, N.J.) was used to detect and measure spontaneous excitatorypostsynaptic potential (sEPSP) events with a minimal amplitude of 0.2 mVand minimal area of 3 mV*msec. See Chakroborty et al., 2012, J.Neurosci. 32(24):8341-53 for further experimental details.

Ca² signaling was measured in individual fura-2 filled pyramidal neuronsvia 2-photon imaging as above, except that bath aCSF was constantlyperfused and caffeine (10 mM for 1 minute) was applied through bathperfusion. Voltage-gated Ca²⁺ channel (VGCC) responses were elicited bydepolarization via current injected through the patch pipette with thecurrent level adjusted to elicit a train of 8-10 spikes. VGCC Ca²⁺responses were measured 3-4 minutes prior to the caffeine RyR Ca²⁺measurement, and each slice was discarded after exposure to caffeine.Test inhibitor compounds to be assayed were introduced during a 1 hourpreincubation and maintained in the aCSF perfusion before, during andafter caffeine application.

Traumatic Brain Injury Induction.

Mice are deeply anesthetized with isoflurane, and have a smallcraniotomy performed over the sensorimotor cortex (3×3 mm) and then aregiven a single CCI (controlled cortical impact) with settings to producemild TBI using the Benchmark Impactor Stereotaxic TBI device. Thecortical contusion injury is administered using modified proceduresdeveloped previously (Sutton et al., 1993, J. Neurotrauma 10(2):135-49).The injury is produced by a flat and circular impactor tip (2 mmdiameter). The impactor is angled 18.0 degrees away from vertical whichenables the flat impactor tip to be perpendicular to the surface of thebrain at the site of injury. Once in place, the impactor tip penetratesthe exposed brain at 3.0 m/s at a depth of 0.6 mm below the corticalsurface based on a stereotactic mouse brain atlas The injury isdelivered unilaterally and the extent of cortical injury will be definedby changes in cytoarchitectural distribution following CCI. After theprocedure, the scalp is sutured closed with monofilament nylon suturesusing a continuous suture or an interrupted pattern and treated withlidocaine. At either 1, 7 or 30 days post TBI induction, mice aretranscardially perfused, and the brains fixed with 4% paraformaldehyde.Subsequent immunostaining using standard protocols to measurephospho-tau species and amyloid species are performed in saline treatedand drug treated mice.

Transcardial Perfusions.

Mice are deeply anesthetized with urethane, the thoracic cavity opened,and cannula inserted into the left ventricle of the heart, and a smallhole cut into the right atrium. Ice cold saline (3 mL) and then 4%paraformaldehyde (5 mL), or 4% paraformaldehyde/1% glutaraldehyde areperfused through the circulatory system. Following this, the mice aredecapitated with sharp scissors and the brain prepared for staining ormicroscopy.

Cell Culture and Dye Loading.

N2a cells (ATCC #CCL-131) were cultured in 50:50 Dulbecco's modifiedEagle's medium: Opti-modified Eagle's medium (Gibco 11995-065:Gibco31985-070), 5% Fetal Bovine Serum (Gibco 26140-079), 1% ABAM (Gibco15240), and incubated at 37° C. with 10% CO₂. Cells were passaged using0.05% Trypsin-EDTA (Gibco 25300-062) for 5 minutes at room temperaturefor non-mechanical dissociation from plate. Enzyme action was haltedwith 10% FBS in DMEM and cells plated onto Poly-L coated round glassslip covers (Chemglass CLS-1760-012) at 30% confluency and then grownovernight in 2.5% FBS in 50:50 DMEM:OptiMEM at 37° C. with 10% CO₂. Thefluorescent calcium indicator, fura-2AM (Invitrogen F1201), was dilutedin DMSO to 1 mM. N2a cells were incubated in 5 μM fura-2AM (diluted infresh media) for 30 minutes, then washed in 1×PBS, fresh media was added(50:50), and allowed to de-esterify for a minimum of 15 minutes. 10 μMof dantrolene or test compound was added and incubated for 30 minutes.All incubations and washes were at 37° C. with 10% CO₂.

Ca²⁺ Imaging and Compound Application.

Glass cover slips with fura 2-AM-filled N2a cells were placed in asubmersion chamber on the stage of an upright Olympus BX51 microscopewhich is coupled to a 2-photon laser imaging system. To evoke aRyR-mediated calcium response, caffeine (5 mM) was bath applied via agravity-driven perfusion system to N2a cells incubated (30 minutes) ineither control media, dantrolene, or one of the test compounds (10 μM).Ca²⁺ imaging of individual cells was accomplished using a custom-madevideo-rate 2-photon imaging system. Laser excitation was provided by 100fs pulses at 780 nm (80 MHz) from a Ti:sapphire laser (Mai TaiBroadband, Spectra-Physics). The laser beam was scanned by a resonantgalvanometer (General Scanning Lumonics), allowing rapid (7.9 kHz)bidirectional scanning in the x-axis, and by a conventional lineargalvanometer in the y-axis, to provide a full-frame scan rate of 30frames/s. The laser beam was focused onto the cells through an Olympus40× water-immersion objective (numerical aperture 0.8). Emittedfluorescence light was detected by a wide-field photomultiplier(Electron Tubes) to derive a video signal that was captured and analyzedby Video Savant 5.0 software (10 Industries). Further analysis ofbackground-corrected images was performed using MetaMorph software. Forclarity, results are expressed as inverse ratios so that increases in[Ca²⁺ correspond to increasing ratios. The % change is calculated as[(F₀/ΔF)−1]*100 where F₀ is the average resting fluorescence at baselineand ΔF is the decrease of fluorescence reflecting Ca²⁺ release.Differences between treatment groups were assessed using two-way ANOVAand Scheffe post hoc analysis for significance (p<0.05). Somatic Ca²⁺responses in all cells in the field of view were analyzed (n=8-20), withthe nucleus region excluded. All compounds were tested in triplicate.

Example 2: RyR Stabilization by Dantrolene

The procedures set forth in Example 1 were used to investigate whetheran RyR-stabilizing compound (dantrolene) is capable of affectingneuronal properties relating to AD pathogenesis and traumatic braininjury.

In two AD models (3×Tg-AD and PS1/APP), sub-chronic (4-week) treatment(5-10 mg/kg, consistent with clinical dosages in the human population)with dantrolene (1-{[5-(4-nitrophenyl)-2-furyl]methylideneamino}imidazolidine-2,4-dione; the nanocrystal formulation of dantrolenesodium is branded as RYANODEX®) returned the exaggerated ER-Ca²⁺ releasein dendritic spines to NonTg levels (FIG. 1), restored synaptictransmission properties and synaptic integrity (FIG. 2), significantlyreduced soluble and insoluble Aβ□ deposition (FIG. 2E), and normalizedRyR2 expression (FIG. 3). Not only do these findings support a centralrole for RyR-Ca²⁺ dysregulation in AD pathogenesis and amyloid betaaggregation, they also demonstrate that even at later disease stagestherapeutic effects are obtained, which supports a feed-forward diseasemechanism involving Ca²⁺ dyshomeostasis, amyloid pathology, and synapticdysfunction.

There is also evidence that stabilizing RyR function with dantrolenereduces phosphorylated tau in the TgCRND8 mouse model of AD (FIG. 4) inaddition to the amyloid pathology. Similarly, in mouse models of TBI,dantrolene treatment significantly reduces the amount of phosphorylatedtau staining in the hippocampal dentate gyrus (FIG. 5).

Note that chronic oral treatment (10+ months) with dantrolene has beenfound to increase amyloid pathology (Zhang et al., 2010, Journal ofNeuroscience 30:8566-8580), suggesting a need for RyR2-targetingcompounds that are not as pathogenic as dantrolene.

Example 3: Hit-to-Lead Optimization of RyR2-Stabilizing Compounds

Although initially based on scaffolds analogous to dantrolene,

new and entirely distinct drug-like analogs have been developed thatmaintain robust in vitro activity and display improved drug properties(see FIG. 6). Substructural features were incorporated intodantrolene-like scaffolds to impart improved drug properties, thusenabling the development of systemically active candidates for AD andother neurodegenerative diseases.

Synthesis of the diverse RyR stabilizers was be accomplished withestablished Suzuki reaction chemistry followed by hydrazone formation.Twenty diverse aryl halides and 5 hydrazines were used to generate 100new candidates. Briefly, 5-formyl-2-furylboronic acid was treated witharyl halides (R₁) under Suzuki reaction conditions using catalytictetrakis(triphenylphosphine) palladium(0) in the presence of a base suchas cesium carbonate. The resulting diaryl species was then subject tohydrazone formation using hydrazine derivatives (R₂) in 1% aceticacid/dimethylformamide. The 20 aryl halide R1 derivatives includeddiverse substituted benzenes and heteroaromatic aryl halides. The fiveR2 hydrazines included hydantoin, urea, and amide functionalities aswell as different aryl groups that imparted diverse drug metabolismpharmacokinetics (DMPK) profiles.

Compounds were purified and then tested by HPLC-MS and were >95% pure.Compounds exhibited aqueous solubilities ranging from 15 mg/mL to >200mg/mL at pH 7.4. Specific emphasis was placed on the incorporation ofsolubilizing moieties to improve the aqueous solubility over dantrolene.

Example 4: Structure-Activity Relationships of First-GenerationCompounds

Compounds were screened to determine whether they would undergo furthertesting or would be excluded from further investigation. The screeningcascade included medicinal chemistry assays, rapid throughput screeningtests in cell culture assays, acute brain slice preparations fromnon-transgenic (NonTg) control and AD mice, and efficacy in chronicallytreated AD mice. Methods for these tests are discussed in Example 1.Lead compounds were effective in N2A cells which predominantly expressthe RyR2 isoform.

Compounds selected after screening are identified in Table 1 and FIG.10, which also present structure-activity relationship information forthe compounds.

TABLE 1 Structure-activity relationships for compounds selected from theinitial round of synthesis.

Log Ca²⁺ Brain/ R₁ R₂ R₃ R₄ R₅ MW P Inc Plasma Caffeine — — — — — — —3.09 — (+con) dantrolene H H NO₂ H

314 1.7 1.96 <0.01 RD05 H H H H

269 1.8 1.43 0.5 RD09 H NO₂ H H

273 2.5 1.06 nd RD10 Cl H NO₂ H

349 3.4 4.25 nd RD11 H NO₂ H H

336 3.0 1.38 nd RD14 CH₃ CH₃ NO₂ H

302 3.2 2.38 1.6 RD54 Cl H NO₂ H

385 5.1 >6 nd RD77 H H Br H

414 5.0 >6 nd RD83 H H NO₂ H

404 5.5 >6 nd RD91 Cl Cl H H

360 4.4 >6 nd RD95 OCH₃ H H NO₂

304 2.5 2.11 nd

Several analogs effectively block RyR-evoked Ca²⁺ in N2A cells whentested at 10 μM (Table 1). Dantrolene reduces caffeine's 3.09-fold Ca²⁺increase over baseline to 1.96, and also shows efficacy in the mousemodel of AD. Thus, it is reasonable to conclude that analogs that show asimilar or better reduction are considered active. Specifically, RD09,RD11, and RD05 block calcium release to a greater degree thandantrolene, and RD95 and RD14 are comparable to dantrolene's effect oncaffeine-stimulated Ca²⁺ release.

A nitro substituent on the left-hand phenyl ring appears to be importantfor activity as dantrolene and the most active compounds, RD09 and RD11,have this functionality. However, nitro groups are associated withreduced solubility and bioavailability. Remarkably and unexpectedly,RD05 does not possess the nitro group, yet retains all activitynonetheless. Also unexpectedly, the brain levels of RD05 are >50 timesthose of dantrolene. These substitutions of functional groups impartadditional metabolic stability and solubility, increasing overallbioavailability compared to dantrolene.

Substitution of the hydantoin functionality of dantrolene was alsoexplored. In several compounds, this functional group was replaced withacetyl and urea features, and the resulting compounds retained activity(for example, compounds RD09, RD95, and RD14). Aromatic features werealso introduced in place of the hydantoin group of dantrolene, as incompound RD11, which add to improvements in overall DMPK as theionizable pyridyl moiety can be transformed into various salts.

Synthetic methods for the compounds shown in Table 1 are as follows:

RD05.

5-Phenyl-2-furaldehyde (1.0 mmol) is dissolved in dimethylformamide (2mL). This solution is added dropwise to a solution of3-aminoimidazolidine-2,4-dione (1.0 mmol) and 1.0 M hydrochloric acidsolution (260 μL) in dimethylformamide (2 mL). After addition iscomplete, the mixture is stirred for 25 h at rt. The mixture is dilutedwith water and extracted with 2 volumes of dichloromethane. The organiclayers are collected and the solvent is removed by rotary evaporation.The residue is purified by preparative reverse-phase HPLC using awater-acetonitrile gradient to afford the desired product. ESI-MS: m/z270 [M+H]+. RD05 is also referred to as SM009.

RD09.

5-(3-Nitrophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof acetic hydrazide (1.0 mmol) and 1.0 M hydrochloric acid solution (260μL) in dimethylformamide (2 mL). After addition is complete, the mixtureis stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 274 [M+H]⁺.

RD10.

5-(2-Chloro-4-nitrophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof 4-N-methyl-N-1-piperazinecarbohydrazide (1.0 mmol) and 1.0 Mhydrochloric acid solution (260 μL) in dimethylformamide (2 mL). Afteraddition is complete, the mixture is stirred for 25 h at rt. The mixtureis diluted with water and extracted with 2 volumes of dichloromethane.The organic layers are collected and the solvent is removed by rotaryevaporation. The residue is purified by preparative reverse-phase HPLCusing a water-acetonitrile gradient to afford the desired product.ESI-MS: m/z 350 [M+H]⁺.

RD11.

5-(3-Nitrophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof 2-pyridinecarbohydrazide (1.0 mmol) and 1.0 M hydrochloric acidsolution (260 μL) in dimethylformamide (2 mL). After addition iscomplete, the mixture is stirred for 25 h at rt. The mixture is dilutedwith water and extracted with 2 volumes of dichloromethane. The organiclayers are collected and the solvent is removed by rotary evaporation.The residue is purified by preparative reverse-phase HPLC using awater-acetonitrile gradient to afford the desired product. ESI-MS: m/z337 [M+H]⁺.

RD14.

5-{4-Nitro-2,3-dimethylphenyl}-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof semicarbazide (1.0 mmol) and 1.0 M hydrochloric acid solution (260μL) in dimethylformamide (2 mL). After addition is complete, the mixtureis stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 303 [M+H]⁺.

RD54.

5-(2-Chloro-4-nitrophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof N-phenylhydrazinecarboxamide (1.0 mmol) and 1.0 M hydrochloric acidsolution (260 μL) in dimethylformamide (2 mL). After addition iscomplete, the mixture is stirred for 25 h at rt. The mixture is dilutedwith water and extracted with 2 volumes of dichloromethane. The organiclayers are collected and the solvent is removed by rotary evaporation.The residue is purified by preparative reverse-phase HPLC using awater-acetonitrile gradient to afford the desired product. ESI-MS: m/z386 [M+H]⁺.

RD77.

5-(4-Bromophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof 4-nitrobenzhydrazide (1.0 mmol) and 1.0 M hydrochloric acid solution(260 μL) in dimethylformamide (2 mL). After addition is complete, themixture is stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 415 [M+H]⁺.

RD83.

5-(4-Nitrophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof 2,4-dichlorobenzhydrazide (1.0 mmol) and 1.0 M hydrochloric acidsolution (260 μL) in dimethylformamide (2 mL). After addition iscomplete, the mixture is stirred for 25 h at rt. The mixture is dilutedwith water and extracted with 2 volumes of dichloromethane. The organiclayers are collected and the solvent is removed by rotary evaporation.The residue is purified by preparative reverse-phase HPLC using awater-acetonitrile gradient to afford the desired product. ESI-MS: m/z405 [M+H]⁺.

RD91.

5-(2,3-Dichlorophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof 2-pyridinecarbohydrazide (1.0 mmol) and 1.0 M hydrochloric acidsolution (260 μL) in dimethylformamide (2 mL). After addition iscomplete, the mixture is stirred for 25 h at rt. The mixture is dilutedwith water and extracted with 2 volumes of dichloromethane. The organiclayers are collected and the solvent is removed by rotary evaporation.The residue is purified by preparative reverse-phase HPLC using awater-acetonitrile gradient to afford the desired product. ESI-MS: m/z361 [M+H]⁺.

RD95.

5-(2-Methoxyphenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof semicarbazide (1.0 mmol) and 1.0 M hydrochloric acid solution (260μL) in dimethylformamide (2 mL). After addition is complete, the mixtureis stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 260 [M+H]⁺.

Example 5: Synthesis of Second-Generation Compounds

Medicinal chemistry optimization was done to provide compounds that arepotent, selective, and systemically available in vivo. There have beenrelatively few efforts focusing on optimization of the in vivo profilesof RyR stabilizers, specifically pharmacokinetic (PK) parameters andefforts to increase brain exposure when administered systemically invivo. Thus, second-generation synthetic efforts were focused ondeveloping RyR stabilizers that possess an ideal ensemble of properties.

The synthetic chemistry strategy was based on the established leadseries pharmacophore, exemplified by compound RD05, which exhibitedsignificant in vitro blockage of calcium release, reasonablebrain-plasma concentrations in vivo, and a potential for efficacy in ourAD mouse models.

Key substructural features necessary for normalizing Ca²⁺ release wereidentified and, using an iterative strategy, >100 analogs weresynthesized that systematically interrogated each region of thepharmacophore to provide optimized candidates having an ensemble ofdesired physical and biological properties. Selected candidates from theoptimization step, along with the lead candidates from thefirst-generation synthesis of compounds described in Example 3, areshown in Table 2.

TABLE 2 Selected compounds after second-generation synthesis. CompoundStructure CK008

CK010

CK011

CK012

CK013

CK017

DL041

RD05

RD09

RD11

RD14

RD95

SM008

Synthetic methods for the compounds shown in Table 2 (and not inTable 1) are as follows:

CK008.

5-(4-Chlorophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof acetic hydrazide (1.0 mmol) and 1.0 M hydrochloric acid solution (260μL) in dimethylformamide (2 mL). After addition is complete, the mixtureis stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 263 [M+H]⁺.

CK0010.

5-(4-Chlorophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof propanohydrazide (1.0 mmol) and 1.0 M hydrochloric acid solution (260μL) in dimethylformamide (2 mL). After addition is complete, the mixtureis stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 277 [M+H]⁺.

CK011.

N′-((5-phenylfuran-2-yl)methylene)propionohydrazide:5-Phenyl-2-furaldehyde (50 mg, 0.29 mmol) is dissolved indimethylformamide (1 mL). This solution is added dropwise to a solutionof propionohydrazide (36.4 mg, 0.41 mmol) and 1.0 M hydrochloric acidsolution in dimethylformamide (1 mL). After addition is complete, themixture is stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford N′-((5-phenylfuran-2-yl)methylene)propionohydrazide.

CK012.

N′-((5-phenylfuran-2-yl)methylene)hydrazinecarbohydrazide:5-Phenyl-2-furaldehyde (50 mg, 0.29 mmol) is dissolved indimethylformamide (1 mL). This solution is added dropwise to a solutionof carbohydrazide (37 mg, 0.41 mmol) and 1.0 M hydrochloric acidsolution in dimethylformamide (1 mL). After addition is complete, themixture is stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to affordN′-((5-phenylfuran-2-yl)methylene)hydrazinecarbohydrazide. ESI-MS: m/z245 [M+H]⁺.

CK013.

5-Phenyl-2-furaldehyde (1.0 mmol) is dissolved in dimethylformamide (2mL). This solution is added dropwise to a solution of phenylacetichydrazide (1.0 mmol) and 1.0 M hydrochloric acid solution (260 μL) indimethylformamide (2 mL). After addition is complete, the mixture isstirred for 25 h at rt. The mixture is diluted with water and extractedwith 2 volumes of dichloromethane. The organic layers are collected andthe solvent is removed by rotary evaporation. The residue is purified bypreparative reverse-phase HPLC using a water-acetonitrile gradient toafford the desired product. ESI-MS: m/z 305 [M+H]⁺.

CK017.

5-(4-Chlorophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof carbohydrazide (0.5 mmol) and 1.0 M hydrochloric acid solution (260μL) in dimethylformamide (2 mL). After addition is complete, the mixtureis stirred for 25 h at rt. The mixture is diluted with water andextracted with 2 volumes of dichloromethane. The organic layers arecollected and the solvent is removed by rotary evaporation. The residueis purified by preparative reverse-phase HPLC using a water-acetonitrilegradient to afford the desired product. ESI-MS: m/z 468 [M+H]⁺.

DL041.

5-(3-Nitrophenyl)-2-furaldehyde (1.0 mmol) is dissolved indimethylformamide (2 mL). This solution is added dropwise to a solutionof phenylacetic hydrazide (1.0 mmol) and 1.0 M hydrochloric acidsolution (260 μL) in dimethylformamide (2 mL). After addition iscomplete, the mixture is stirred for 25 h at rt. The mixture is dilutedwith water and extracted with 2 volumes of dichloromethane. The organiclayers are collected and the solvent is removed by rotary evaporation.The residue is purified by preparative reverse-phase HPLC using awater-acetonitrile gradient to afford the desired product. ESI-MS: m/z350 [M+H]⁺.

SM008.

5-Phenyl-2-furaldehyde (1.0 mmol) is dissolved in dimethylformamide (2mL). This solution is added dropwise to a solution of 1-phenylethylamine(1.0 mmol) and 1.0 M hydrochloric acid solution (260 μL) indimethylformamide (2 mL). After addition is complete, the mixture isstirred for 25 h at rt. The mixture is diluted with water and extractedwith 2 volumes of dichloromethane. The organic layers are collected andthe solvent is removed by rotary evaporation. The residue is purified bypreparative reverse-phase HPLC using a water-acetonitrile gradient toafford the desired product. ESI-MS: m/z 275 [M+H]⁺.

Example 6: Rapid Screening Assay in Cell Culture Systems and Neuronsfrom AD Mice

The RyR2-stabilizing compounds presented in Example 5 were tested usinga RyR2-expressing N2A cell line, grown on 96 well-plates, and tested ona high speed automated imaging system as disclosed in Example 1.Compounds were screened for the ability to reduce RyR-evoked calcium inresponse to strong agonist stimulation, as similarly demonstrated withdantrolene (FIG. 7).

Compounds were also tested in hippocampal brain slice preparations fromnon-transgenic (NonTg) and AD (PS1/APP) mice as disclosed in Example 1to determine their ability to normalize aberrant RyR-modulated calciumsignals evoked by physiological stimulation in AD mice. As shown in FIG.8A, as with dantrolene, treatment with CK013 and CK017 normalizedaberrant RyR-evoked calcium signals. Treatment with all of the compoundstested had minimal effects on control NonTg mice, nor did it affectother fundamental Ca²⁺ signaling events not altered in the AD mice, suchas voltage-gated Ca²⁺ channel influx, passive membrane properties, orspiking properties (FIGS. 8B-8D).

Example 7: In Vivo Studies

Compounds were tested with an in vivo sub-chronic treatment paradigm inNonTg and AD mice to determine if aberrant RyR-calcium responses arenormalized in the AD mice while not impinging on distinct calciumpathways unaffected in AD.

Ca²⁺ signaling and electrophysiological properties of CA1 pyramidalneurons were studied in hippocampal brain slices according to theprotocols disclosed in Example 1. Briefly, 3×Tg-AD mice were treatedwith either saline (0.9%), CK013 or CK017 for four weeks (10 mg/kg; ip),and then acute hippocampal brain slice preparations were made for thepurposes of performing whole cell patch clamp recordings and 2-photoncalcium imaging in CA1 pyramidal neurons. The objective was to determineif our compounds could restore normal RyR-mediated calcium signaling inthe AD mice and maintain normal membrane physiology. The results, shownin FIG. 9, demonstrate that in both the CK013 and CK017 treated 3×Tg-ADmice, the RyR-evoked calcium responses are not different from the NonTgcontrol responses, and are significantly reduced compared to theaberrant RyR-calcium responses in the saline treated 3×Tg-AD mice.

Also, passive membrane properties such as resting membrane potential andmembrane input resistance were not affected by these compounds. As shownin Table 3, there were no statistically significant differences inresting membrane potential (RMP) and membrane input resistance (Ri)among the groups (p>0.05). Additionally, there were no statisticallysignificant differences in the ratio of heart weight:body weight amongthe treatment groups (Table 4; p>0.05), nor were there differences inabsolute body weights or heart weights among the groups (data not shown;p<0.05).

TABLE 3 Passive membrane properties from CA1 neurons. Strain/Treatment(n = neurons) RMP (mV) Ri NonTg Saline (10)   −72 ± 0.1 154 ± 11 3xTg-ADSaline (5)   −70 ± 0.4 157 ± 12 3xTg-AD CK013 (15) −69.5 ± 0.4 152 ± 203xTg-AD CK017 (14) −69.0 ± 0.2 144 ± 13

TABLE 4 Ratio of heart weight:body weight after treatment withRyR-stabilizers. Strain/Treatment (n = animals) Ratio of heartweight:body weight NonTg Saline (4)  4.4 × 10⁻³ 3xTg-AD Saline (4) 4.05× 10⁻³ 3xTg-AD CK013 (4) 4.06 × 10⁻³ 3xTg-AD CK017 (4)  3.6 × 10⁻³

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein asparticularly advantageous, it is contemplated that the present inventionis not necessarily limited to these particular aspects of the invention.

We claim:
 1. A method for normalizing neuronal calcium dyshomeostasis ina subject in need thereof comprising administering to the subject aneffective amount of the compound selected from the group consisting of:

and a pharmaceutically acceptable salt thereof.
 2. The method of claim1, wherein the subject is a human subject.
 3. A method for treating aneurological or neurodegenerative disorder in a subject, wherein theneurological or neurodegenerative disorder is Alzheimer's disease ortraumatic brain injury, comprising administering to the subject aneffective amount of the compound

and a pharmaceutically acceptable salt thereof.
 4. The method of claim 3wherein the neurological or neurodegenerative disorder is traumaticbrain injury.
 5. The method of claim 3, wherein the neurological orneurodegenerative disorder is Alzheimer's disease.